Understanding the mechanisms of synaptic development will be central to understanding the effects of genetic and environmental factors (including drugs) on the fetus and young child. The development of the nerve-muscle synapse in vivo and in tissue culture is a valuable model of CNS synaptic development. A striking feature of this nerve-muscle synapse development is the formation of an aggregate of nicotinic acetylcholine receptors (AChR) in the postsynaptic membrane. In at least some species, an increase in the rate of insertion of AChRs into the membrane at the site of the nascent synapse makes an important contribution to the formation of this aggregate. A 42kD protein has now been purified (Usdin and Fischbach, 1986) from the chicken brain which, when present at a concentration on the order of 0.5ng/ml, causes an increase in the rate of AChR incorporation into the membrane of cultured chick myotubes and also in the number of AChR aggregates. This protein might be released by the spinal motor neuron at the site of nerve-muscle contact and induce synaptic specializations. The purpose of this project is to investigate the biological role of this protein and its mechanism of action. The specific aims are: 1) Purify enough of the 42kD acetylcholine receptor inducing activity (42kD ARIA) to provide amounts needed for production of antibodies, determination of amino-terminal sequence, and studies of the biological properties of this protein. 2) Raise monoclonal and polyclonal antibodies directed against this 42kD ARIA. 3) Use these antibody reagents to investigate the biological role of this protein. A key question is whether or not antibodies directed against this protein block the formation of synaptic specializations at sites of nerve-muscle contact. This will be investigated in vitro and in vivo. The regional distribution of the 42kD ARIA will be studied using immunofluorescence, and an ELISA assay will be developed to quantitate levels of the 42kD ARIA. The effect of focal application of this protein onto myotubes will also be examined. 4) Long-term goals include determining the full amino acid sequence of the 42kD ARIA and studying its affect on the rate of synthesis of AChR subunits and on subunit mRNA levels.